Proteins perform a vast array of functions within living organisms and are the chief actors within a cell. Examples of such functionality include catalyzing metabolic/chemical reactions (i.e. enzymes), cell signaling and signal transduction, DNA replication, providing structural support (i.e. structural proteins), and transporting molecules from one location to another. Proteins differ from one another primarily with respect to their sequence of amino acids, which is dictated by the nucleotide sequence of their genes and typically results in protein folding into a specific three-dimensional (3D) structure that determines its activity.
In basic ribosomal protein biosynthesis, messenger RNA (mRNA) encodes a protein through the process of translation. In brief, ribosome molecules read down the length of mRNA codons (each a sequence of three-nucleotides) and translate the genetic information contained therein to a specific sequence of amino acids by facilitating complementary base pairing to the complementary transfer RNA (tRNA) anticodons.
Each mRNA codon is recognized by a particular tRNA. During translation, each of the tRNA molecules that bind the mRNA is “charged,” meaning that it is carrying a specific amino acid via a covalent bond. As such, when a particular tRNA binds with its complementary codon on the mRNA at the ribosome, its cargo amino acid is lined up with the amino acid of the tRNA corresponding to the next codon on the mRNA. Thereafter, a peptide bond forms between the amino acids and the tRNA releases its amino acid, thus forming a chain of amino acids—or a polypeptide—as the mRNA pass through and are read by the ribosome. Termination of the polypeptide happens when the ribosome hits a stop codon in the mRNA that ends the translation process. The polypeptide is released and folds into its dictated 3D geometry. Accordingly, the specific nucleotide sequence of an mRNA specifies which amino acids are incorporated into the protein product, with the role of tRNA being to specify which amino acids correspond with the sequence from the mRNA. The particular sequence of amino acid in a protein product has a direct effect as to the resulting structure and, thus, functionality of the protein.
Conventionally, each type of tRNA molecule can be attached to only one type of amino acid. The covalent attachment between the tRNAs and their specific amino acids is catalyzed by aminoacyl tRNA synthetases. Typically, aminoacyl tRNA synthetases are extremely specific with respect to tRNA and the related amino acid. Accordingly, a specific tRNA substrate will only take on a particular amino acid when aminoacylated with the correct aminoacyl tRNA synthetase
Through protein engineering techniques, the natural translation process can be manipulated to study protein structure and function, as well as for protein modification. Indeed, protein engineering has become an extensively used tool in molecular biology, with methods for incorporating even unnatural amino acids (uAAs) into proteins to develop unique functionalities and/or improved protein function. For example, the site-specific introduction of uAAs can be used to probe enzyme mechanisms, increase acidity, localize proteins within cells (through adding a fluorescent label or otherwise), improve the therapeutic properties of drugs, and the like. Because of the potential in this area, there is significant interest in expanding the chemical diversity of proteins beyond the twenty (20) amino acids most commonly incorporated during ribosomal protein synthesis.
The most common conventional method of introducing an uAA during protein biosynthesis employs a functional pair of tRNA and aminoacyl tRNA synthetase (“ARS”)—an orthogonal set—to act independently of the endogenous aminoacylation machinery of the cell. Specifically, the ARS is engineered to charge a tRNA (for example, an amber suppressor tRNA) with the particular uAA of interest while the tRNA recognizes a specialized codon within the mRNA (typically an amber STOP codon (UAG, for example) or a four-base codon) that does not code for one of the natural amino acids. Accordingly, the charged tRNA delivers the uAA to the ribosome for protein synthesis and, in doing so, uniquely introduces the uAA into a protein at the desired site.
To be introduced selectively at its predetermined position only, the orthogonal set must not crosstalk with the endogenous tRNA and synthetase sets, while remaining functionally compatible with the ribosome and other components of the translation apparatus. This is problematic for several reasons, one of which being that many tRNA synthetases recognize the anticodon loop of the tRNA and thus cannot be used to charge a tRNA that recognizes a stop codon. Furthermore, the active site of the ARS must be capable of accommodating the uAA of interest. As proteins normally have exquisite specificity for their substrates, this can significantly limit the identity of the uAA accepted.
Orthogonal tRNA synthetases are conventionally generated using archaeal proteins that, when introduced into prokaryotes such as E. coli, can discriminate between all of the tRNA substrates available in a cell or in an in vitro translation system and bind only to its orthogonal tRNA partner. In this manner, only the appropriate tRNA substrate is charged with the uAA. However, Eukarya is the most complex domain of life, with not only more tRNA gene content, but also higher variation. So while conventional orthogonal methodologies have had some success in prokaryotic and in vitro translation systems, a robust mechanism to introduce uAAs into eukaryotes has yet to be established. Indeed, the foreign ARS and tRNA conventional pairs are typically not successful when used in vivo in eukaryotic cells, as there is a substantial risk the orthogonal set will recognize—and charge—their homologs derived from the host organism. Furthermore, as previously stated, significant engineering is required to redesign the orthogonal ARS such that it will accept the uAA, and each desired modification within a protein potentially requires additional engineering of the ARS.
Accordingly, what is needed is an efficient and effective system capable of selecting and selectively charging tRNA with a wide variety of uAAs within a eukaryotic cell.